Microspheres are utilized widely for biological assays. Fluorescent dyes can be utilized for multiplexing and identification of microspheres from one another. It is well known that fluorescent dye from these microspheres leaches out from the microspheres over time. This leads to a decrease in fluorescent intensity as well as an increase in background fluorescence. Loss of accuracy, especially in measurement of MESF (Molecules of Equivalent Soluble Fluorochrome), is undesirable. Loss of fluorescence over time can change the quantitative level of fluorescent dye in the microspheres and therefore lead to errors.
One standard method of staining microspheres is by swelling the particles and incorporating the fluorescent dye in a non-covalent manner. This leads to dye leaching over time. For instance, an oil-soluble dye in solvent mixed with copolymer particles can be utilized with hydrophobic microspheres. During the swelling process, the microparticles have increased pore sizes, thus allowing permeating of fluorescent dye into the matrix. The solvent type, time, and temperature controls the level of swelling. Removal of the solvent reverses the process and traps the fluorescent dye in the matrix. This mixing leads to staining of the microspheres, but there is tendency of the dyes to leach out over time. Although there are hydrophobic type interactions, the use of amphiphilic detergents in bead mixtures facilitates this leaching process.
Methods to covalently attach fluorophores to the surface of polymeric microspheres are also known. Furthermore, the abundance of commercially-available microspheres makes this approach standard to one skilled in the art. However, surface labeling is not desirable because only a limited amount of fluorescent dye can be incorporated in this manner.
It is therefore highly desirable to have microparticles for bioassay purposes that have fluorescent molecules that are stable and do not leach out over time, permitting these microparticles to be identified by their fluorescence signature.